Modulation of animal cellular responses with compositions containing isoxanthopterin-8-(1&#39;-β-aldoglycosidyl) derivatives

ABSTRACT

Animal cellular responses, and particularly immune-related responses, are modulated by contacting such cells with a unit dose of a composition containing an effective amount of an isoxanthopterin-8-aldoglycoside. An isoxanthopterin has a structure that conforms to the formula ##STR1## wherein R 1  is a substituent and R 2  is an aldoglycoside.

CROSS-REFERENCE TO COPENDING APPLICATION

This is a continuation-in-part of copending patent application Ser. No.808,886 filed Dec. 13, 1985, which is a continuation-in-part of Ser. No.798,629, filed Nov. 15, 1985, now U.S. Pat. No. 4746651, which is itselfa continuation of copending patent application Ser. No. 546,679, filedNov. 1, 1983, now U.S. Pat. No. 4,643,992.

TECHNICAL FIELD

The present invention relates to modulation of animal cellularresponses, and more particularly, to modulation of antigen-specificimmune and other animal cellular responses by compositions containing alow molecular weight derivative of isoxanthopterin.

BACKGROUND ART

An animal's immune system is comprised of numerous elements that actseparately and/or in concert to counteract, to eliminate, or toneutralize substances that are recognized by that system as foreign tothe animal host. Generally, but not necessarily, the substancerecognized as foreign by the immune system has its origin exogenous tothe host. Exemplary of such exogenous substances are infectious bacteriaand the by-products of their cellular activity, virus particles andtheir proteins, proteins injected by insect stings, and the like. Inautoimmune diseases, such as rheumatoid arthritis, the host's immunesystem perceives host-made proteins or self-made proteins as foreign.

The principal effectors of the immune system are the leukocytes, whichinclude lymphocytes of thymic origin (T cells), lymphocytes produced inbone marrow (B cells), neutrophils which, inter alia, produce enzymesthat make oxidizing agents such as hydrogen peroxide that have cytotoxiceffects upon bacteria, and macrophages, which present the foreignsubstance or immunogen (antigen) to the T cells and B cells, as well asproduce a protein designated interleuken-1 that assists T celltransformation into T helper cells and B cell and T cell proliferation.Complement, which is a complex mixture of proteins that acts in anordered, cascading manner upon the foreign substance, also plays a majorrole in immune responses.

B cells can be distinguished from T cells, inter alia, by the presenceof monomeric immunoglobulins (antibodies) on their surface membranes.Mature B cells secrete antibodies into their environment when properlyactivated.

There are five known classes of immunoglobulins, identified as IgA, IgD,IgE, IgG, and IgM on the basis of five antigenically different heavychain proteins that make up a portion of the immunoglobulin molecule. Bcells also bear non-immunoglobulin cell markers, including a complementreceptor (CR), a receptor for the Fc portion of immunoglobulin (FcR),I-region associated antigens (Ia), and a set of differentiation antigens(Lyb 1-7) that are identified by antisera and other means are correlatedwith various aspects of B cell maturation and activation. These markersare useful in phenotypically identifying B cells and B cellsubpopulations.

While the immunoglobulins act upon the foreign substances, or antigen,the T cells, and particularly helper T cells, are believed necessary tostimulate B cells to divide and to differentiate into antibody secretingcells for humoral immunity. Suppressor T cells contribute to theregulation of humoral immunity, while cytotoxic T cells and T cellmediators of delayed-type hypersensitivity are the principal effectorsof cell-mediated immunity.

Murine T cells bear surface antigens designated Lyt 1, 2, and 3 as wellas L3T4 that are related to T cell functions. Helper T cell precursorsare of the Lyt 1⁺, 2⁻,3⁻ L3T4⁴ phenotype. These cells normallyparticipate in the activation and regulation of B cells.

Helper T cells are known to assist in activation and differentiation ofimmunoglobulin-secreting B cells after a first message is received bythe B cells from the activating immunogenic (antigenic) agent usuallypresented to it after processing by an antigen-presenting cell. However,the mode by which the T cells provide help for activation anddifferentiation of the B cells is a matter of controversy.

The immune response exhibited by animal cells can be modified byartificial suppression (immunosuppression) or enhancement(immunopotentiation). Artifically induced immunosuppression can beachieved by six general methods: (1) administration of a suppressivedose of antigen, (2) administration of specific antisera or antibodies,(3) use of other biologic reagents such as antilymphocyte antisera, (4)use of drugs or hormones, (5) radiation, and (6) surgical removal oflymphoid tissue. Immunopotentiation can be achieved by administration ofan agent effecting (1) an increase in the rate at which the immuneresponse develops, (2) an increase in the intensity or level of theresponse, (3) a prolongation of the response, or (4) the development ofa response to an otherwise non-immunogenic substance.

The agents that are known to enhance immune responses are generallytermed adjuvants and can be placed into two general categories: (1)those providing general potentiation; i.e., substances that enhancecellular and/or humoral immune responses for a wide variety of antigens,and (2) those providing specific potentiation; i.e., substances thatenhance specific responses only to certain antigens.

Substances that can act as adjuvants can be grouped into the followingcategories: (1) water and oil emulsions, e.g., Freund's adjuvant, (2)synthetic polynucleotides and other polyanions, (3) hormones, drugs andcyclic nucleotides, (4) microbial products, e.g., endotoxins, (5)lymphokines and monokines such as the interleukins, and (6) syntheticpeptides, e.g., bestatin and tuftsin.

A substance capable of specifically potentiating the immune response istransfer factor, a dialyzable leukocyte extract (DLE) obtained fromhuman peripheral leukocytes. It has been reported that the transferfactor exhibits some effectiveness in patients with immunodeficienciesand possible effectiveness in cancer patients and in patients withlimited immunodeficiencies. However, the efficacy of this agent ishighly controversial, and much remains to be learned about it.

In some diseases and physiological conditions such as Xchromosome-linked agammaglobulinemias, senescence anddrug-induced-immunosuppression, B cell activation and differentiation islacking or exists only at a reduced level, thereby lessening the immuneresponse capabilities of the host. These diseases and conditions arerepresentative of immunosuppressed states. Here, enhanced B cellactivation and differentiation, if it can be effected, tends tobeneficially lessen the immunological deficits that can manifestthemselves as disease and/or improve the patient's condition.

An immunopotentiated state can be illustrated by the bodily conditionafter vaccination. Here, the immune response is first enhanced due to aprimary response to the vaccine's immunogen, and usually can bebeneficially enhanced still further by a "booster" injection of theimmunogen or vaccine, administered later to provide an improved degreeand/or duration of immunity.

Lymphokines and monokines are immunopotentiating proteins produced bylymphocytes and cells of the monocyte-macrophage lineage, respectively.One monokine, interleukin-1, is produced by macrophages when they arestimulated by a mitogen or antigen. Interleukin-1 is usually requiredfor producing a primary antigenic response.

Interleukin-1 assists in the production of interleukin-2 by T cells.Interleukin-2 is a growth factor for T cells and assists in thetransformation of helper T cells. Thus, induction of interleukin-1production or of a protein-responsive activity or T cells similar tothat produced by interleukin-1 would be beneficial in enhancing immuneresponses, particularly where macrophages are absent or where theirproduction of monokines is deficient.

Co-assigned U.S. Pat. No. 4,539,205 to Goodman and Weigle describesmodulation of animal cellular responses with 8-substituted guaninederivatives bonded 9-1' to an aldose having 5 or 6 carbon atoms in thealdose chain (ring). The cellular modulations described in that patentrelate mostly to immunomodulation such as adjuvanticity in producingprimary and secondary immune responses. Activity against certainneoplastic conditions is also disclosed as are T cell-replacingactivity, an IL-1 like activity on thymocytes, and induction of therelease of lysosomal enzymes from neutrophils. The 8-substituents inthose molecules have electron withdrawing inductive effects relative tohydrogen. Thus, halo, mercapto or its thioxo tautomer, acyl mercapto,alkyl sulfido, nitro, cyano, keto, halomethyl and methyleneoxy alkyl andthe like were disclosed as useful, while electron donating substituentssuch as an amino group were found to be inactive.

In addition, co-assigned, co-pending U.S. patent application Ser. No.546,679 and its corresponding published European patent application No.83306791.1 further discloses the use of derivatives of 8-hydroxyguanine(8-oxoguanine), 7-methyl-8-oxoguanine and 7-methyl-8-thioxo-guanine inmodulating animal cellular responses. Further results using guaninederivatives disclosed in U.S. Pat. No. 4,539,205 are also disclosed asare similar results using guanine derivatives disclosed for the firsttime in that application.

U.S. Pat. No. 3,798,210 to Pfleiderer describes the synthesis of8-(1'-glycosidyl)pteridines, including isoxanthopterin derivatives. Thatpatent teaches the use of its compounds as the active pharmaceuticalagents against specific pathogens such as malaria and tubercle bacilli,pathogenic fungi, gram-positive and gram-negative bacteria, andprimarily against viruses such as herpes virus and influenza virus. Someof the compounds of the Pfleiderer patent are also useful herein, butnot as antibiotics as is taught in Pfleiderer. This use is describedhereinafter.

BRIEF SUMMARY OF THE INVENTION

Animal cellular responses are modulated by contacting animal cells witha composition containing a diluent amount of a physiologically tolerablecarrier admixed with an effective amount of an active ingredient that isan isoxanthopterin derivative. The structure of the isoxanthopterinderivative conforms to that of the formula ##STR2##

wherein

R₁ is a radical selected from the group consisting of hydrogen, loweralkyl, hydroxy lower alkyl, polyhydroxy lower alkyl, phenyl,phenyl-lower alkyl, lower alkyl phenyl, lower alkoxy phenyl, halophenyl,trifluoromethyl phenyl, hydroxy, oxo (0═), lower alkoxy, phenyl-loweralkoxy, halo, mercapto, thioxo (═), lower alkylthio, lower alkyloylthio,phenyl-lower alkylthio, lower alkanoyl (lower acyl), carboxy, loweralkoxy carbonyl, lower alkylcarboxy, lower alkylene loweralkylcarboxylate, lower alkoxy lower alkyl carbonyl, and carboxamido andlower alkyl carboxamido in which the carboxamido group has the formulaCONR₃ R₄ wherein R₃ and R₄ are the same or different and are selectedfrom the group consisting of hydrogen and lower alkyl or NR₃ R₄ togetherform a heterocyclic ring having five or six atoms in the ring;

R₂ is a beta-bonded aldoglycoside radical selected from the groupconsisting of 1'-aldopentosidyl, 1'-aldohexosidyl, mono-deoxygenated1'-aldopentosidyl, and mono-deoxygenated 1'-aldohexosidyl and theirO-substituted lower alkyl, lower alkanoyl, benzyl and benzoylderivatives wherein an O-substituent, if present on one oxygen, ispresent on all available ring substituent oxygens;

the pharmaceutically acceptable salts of the isoxanthopterin derivative;and

the tautomers of the isoxanthopterin derivative.

The contact between the cells and the composition is maintained for aperiod of time sufficient for modulation of the contacted cells'responses.

Enhancement of immunogen(antigen)-specific humoral immune responsesresulting in adjuvanticity that provides enhanced antibody secretion inthe presence of immunogen is a particular example of the animal cellularresponse than can be modulated in accordance with the present invention.The term "modulate" in its various grammatical forms, as used herein,designates enhancement as well as inhibition of an animal cellularresponse in vitro and/or in vivo.

A cellular response-modulating composition of this invention can be usedto provoke differing, although related results depending, inter alia,upon the manner of administration, dosage and the cell population towhich it is administered. The active ingredient isoxanthopterinderivative can be present in the composition admixed in the carrier as asuspension of solid isoxanthopterin derivative in a solid or liquidcarrier, or as a dissolved solute in the carrier.

Contacting leukocytes such as B lymphocytes with a composition of thisinvention and maintaining that contact for a predetermined period oftime modulates the immune response of those leukocytes. Modulation of Blymphocyte (B cell) responses can be effected by treating B cells withan effective amount of the immunogen to form immunogen-primed B cells,followed by contacting the B cells with the immune response-modulatingcomposition and a further effective amount of immunogen. B cell immuneresponses can also be modulated by contacting the B cells with a primingimmunogen and an immune response-modulating composition of thisinvention followed thereafter by contacting the immunogen primed cellswith an additional effective amount of the immunogen alone, or with afurther amount of immune response-modulating composition. In addition,an immune response-modulating composition can be administered to contactthe animal cells and thereafter followed, while the isoxanthopterinderivative is in contact with the animal cells; i.e., present in vivo orin vitro, with one or more immunizing doses of an immunogen. These cellresponse modulations are within those effects referred to asadjuvanticity; i.e., the isoxanthopterin derivative acts as an adjuvantfor the immunogen, and thus provides an immunogen- or antigen-specificmodulation.

The methods of this invention can be used on cells in vivo as well as invitro. The compositions can be administered subcutaneously,intravenously intraperitoneally in a liquid form, or perorally as inpill or capsule form, or in liquid form as a slurry, suspension orsolution.

The present invention has several benefits and advantages.

One of the benefits of this invention is that its use can provide the"second message" required for B lymphocyte activation anddifferentiation in response to a first, priming (immunogenic) message.

An advantage of this invention is that contacting animal cells asdescribed herein can lead to the activation and differentiation of thosecells, which in turn can lead to the induction of protein production, asin the case of immunoglobulin (antibody) secretion from B cells,monokine secretion from macrophages, and lymphokine secretion from Tcells.

Another advantage of the present invention is that enhanced immuneresponses can be effected in both the presence and absence of T helpercell activity. Thus, enhanced immune responses are noted in both Tcell-dependent and T cell-independent systems, making this inventionuseful when host leukocytes are immunocompromised from having lost Thelper cell function, as well as in leukocytes with normal T helperfunction.

Still further benefits and advantages of the present invention will beapparent to those skilled in the art from the Detailed Description thatfollows.

DETAILED DESCRIPTION OF THE INVENTION A.Isoxanthopterin-8-Aldoglycosides

2-Amino-4-hydroxypteridine and its derivatives are known in the art aspterin and its derivatives, respectively. Prototropically active pterinsare usually represented in their most favored tautomeric formula as2aminopterin-4-one and its derivatives. Pfleiderer, Chapter 2.16 inComprehensive Heterocyclic Chemistry, Vol. 3, Part 2B, Katritzky andRees eds., Pergamon Press, New York (1984) pages 63-327.

2-Amino-4,7-dihydroxypteridine and its tautomer 2-aminopterin-4,7-dioneare known as isoxanthopterin. A more precise chemical name forisoxanthopterin is 2-amino-3,4,7,8-tethydro-4,7-dioxopteridine. Thecompounds useful herein will generally be referred to as isoxanthopterinand its derivatives. These useful isoxanthopterin derivatives allpossess an aldoglycoside (sugar aldehyde) as a substituent at the8-position of the pteridine ring system, and can also include asubstituent other than hydrogen at the 6-position.

Isoxanthopterin and 6-substituted isoxanthopterins for preparation ofthe isoxanthopterin derivatives useful herein are themselves readilyprepared by known reactions. In one reaction scheme, a2,5,6-triamino-4-hydroxypyrimidine is reacted with an alpha-keto acid inwhich a substituent beta to the carboxy group forms the R₁ group in thestructural formulas herein. See, Hurst, An Introduction To The ChemistryAnd Biochemistry Of Pyrimidines, Purines And Pteridines, John Wiley &Sons, New York, pages 86-103 (1980), and the citations therein. Inanother reaction scheme, the above pyrimidine is reacted with a di-loweralkyl ester of an acetylene dicarboxylic acid to form a lower alkylcarboxylic acid at the 6-position and lower alkyl esters thereof.Iwanami, Bull. Chem. Soc. Japan, 44:1314 (1971). Still further compoundsand reaction schemes are discussed in Pfleiderer, Chapter 2.16 ofComprehensive Heterocyclic Chemistry, supra.

The isoxanthopterin 8-aldoglycoside derivatives useful herein arepreferably prepared from isoxanthopterin or a 6-substitutedisoxanthopterin derivatives to which the aldoglycosidic group isthereafter added by the method of Pfleider as described in U.S. Pat. No.3,798,210, whose disclosures are incorporated herein by reference. Othermethods of preparation such as the cyclization of a2-amino-3,4-dihydro-5-nitro-4-oxo-6-aminoglysidyl-pyrimidine describedby Lohrmann and Forrest, J. Chem. Soc., 460-465 (1965) are also useful.

Briefly, in accordance with the Pfleider technique, a suitablysubstituted isoxanthopterin is O-metalized at the 7-position with aquadrivalent metal of the fourth main group and third to fifth period ofthe periodic system. The O-metalized compound so prepared is reactedwith an aldoglycoside whose 1'-position hydroxyl group is itselfderivatized as a reactive ester such as an ester of a lower carboxylicacid ester like acetic acid, or as an ether such as a lower alkyl etherlike a methyl ether. The 1'-position hydroxyl can also be replaced by ahalo group such as bromide as taught by Pfleider and his co-workers inChem. Ber., 106, 317-331 (1973); Chem. Ber., 106, 1952-1975 (1973); andChem. Ber., 107, 339-361 (1974).

Quadrivalent germanium, tin and especially silicon are preferredO-metalizing agents. The particularly preferred metalizing agent ishexamethyldisilazane.

A strong acid catalyst such as an inorganic acid like sulfuric acid ispreferably used with an O-metalizing agent such as hexamethyldisilazane.The hexamethyldisilazane is preferably utilized in excess, in theabsence of water, and preferably in the presence of nitrogen or argonrather than air.

The 7-O-metalized isoxanthopterin is thereafter typically collected andreacted in an inert solvent such as dry benzene with the aldoglycosidewhose hydroxyl groups other than that of the 1'-position are protected,as by benzoyl or acetyl groups. The 1'-position of the chosenaldoglycoside is protected as before discussed.

The glycosidation reaction is preferably carried out in the presence ofa mercuric salt such as a mercuric halide, or mixture of mercurichalides where an aldoglycosidyl 1'-ether or 1'-ester is used. Anelevated temperature such as that of refluxing benzene at one atmosphereof pressure is used for the aldoglycosylation reaction (condensation ofsugar and isoxanthopterin).

The mercury salt, where used, is filtered from the reaction medium oncethe reaction is over, and the isoxanthopterin-8-(hydroxyprotected)-aldoglycoside) derivative is recovered as by columnchromatography. The hydroxy protecting groups, e.g., benzoyl or acetyl,are thereafter removed by standard procedures such as reaction in sodiummethoxide-methanol, followed by neutralization. The desiredisoxanthopterin-8-(1'-aldoglycoside derivative is thereafter collectedand purified, as by crystallization.

Useful isoxanthoperin derivatives have a structure that corresponds tothe formula ##STR3##

wherein

R₁ is a radical selected from the group consisting of hydrogen, loweralkyl, hydroxy lower alkyl, polyhydroxy lower alkyl, phenyl,phenyl-lower alkyl, lower alkyl phenyl, lower alkoxy phenyl, halophenyl,trifluoromethyl phenyl, hydroxy oxo (O═), lower alkoxy, phenyl-loweralkoxy, halo, mercapto, thioxo (S═), lower alkylthio, loweralkyloylthio, phenyl-lower alkylthio, lower alkanoyl (lower acyl),carboxy, lower alkoxy carbonyl, lower alkylcarboxy, lower alkylene loweralkylcarboxylate, lower alkoxy lower alkyl carbonyl, and carboxamide andlower alkyl carboxamide in which the carboxamido group has the formulaCONR₃ R₄ wherein R₃ and R₄ are the same or different and are selectedfrom the group consisting of hydrogen and lower alkyl or NR₃ R₄ togetherform a heterocyclic ring having five or six atoms in the ring;

R₂ is a beta-bonded aldoglycoside radical selected from the groupconsisting of 1'-aldopentosidyl, 1'-aldohexosidyl, mono-deoxygenated1'-aldopentosidyl, and mono-deoxygenated 1'-aldohexosidyl and theirO-substituted lower alkyl, lower alkanoyl, benzyl and benzoylderivatives wherein an O-substituent, if present on one oxygen, ispresent on all available ring substituent oxygens;

the pharmaceutically acceptable salts of the isoxanthopterin derivative;and

the tautomers of the isoxanthopterin derivative.

Groups and radicals are referred to as "lower" denote that they possess1 to about 6 carbon atoms, and preferably 1 to about 3 carbon atoms.

Lower alkyl radicals include, for example, methyl, ethyl, propyl,iso-propyl, n-butyl, sec-butyl, t-butyl, n-pentyl, 2-methyl-3-butyl,1-methylbutyl, 2-methylbutyl, neo-pentyl, n-hexyl, 1-methylpentyl,3-methylpentyl, 1-ethylbutyl, 2-ethylbutyl, 2-hexyl, 3-hexyl, and thelike.

Hydroxy lower alkyl radicals include hydroxy methyl, 2-hydroxyethyl,2-hydroxypropyl, 3-hydroxypropyl, 3-hydroxy-2-butyl,3-hydroxy-2,2-dimethylpropyl, 6-hydroxyhexyl and the like.

Polyhydroxy lower alkyl radicals include 1,2-dihydroxyethyl,1,2,3-trihydroxypropyl, 2,3-dihydroxypropyl, 3,4-dihydroxybutyl and thelike. Those skilled in the art will understand that the contemplatedpolyols contain no more than one hydroxyl group on each carbon atom ofthe lower alkyl group.

Phenyl-lower alkyl radicals include phenyl-substituted lower alkylradicals listed above, wherein the alkyl position of the radical isbonded to the 6-position of the isoxanthopterin 8-aldoglycoside.Exemplary radicals include benzyl, phenethyl, 2-phenylpropyl,2-phenyl-3-methylpentyl and the like.

Lower alkyl phenyl radicals are the above-described lower alkyl radicalssubstituted on a phenyl radical that is itself bonded to the 6-positionof an isoxanthopterin 8-aldoglycoside. Exemplary of such lower alkylphenyl radicals are o-xylyl, p-(2-hexyl)phenyl, m-(iso-propyl)phenyl,and the like. Trifluoromethylphenyl substituted ortho, meta or para tothe position of binding to the 6-position of the isoxanthopterinconstitute a sub-class of lower alkyl phenyl radicals.

Lower alkoxy phenyl radicals can be viewed as lower alkyl ethers ofortho-, meta- or para-isoxanthopterin substituted phenols, wherein thelower alkyl group is as described before. Exemplary lower alkoxy phenylradicals include o-methoxyphenyl, m-sec-butoxyphenyl, andp-(2-ethylbutoxy)phenyl.

Halophenyl radicals utilize halogen-substituted phenyl radicals in whichthe halogen is preferably fluoro, chloro and bromo, and also includeiodo. Exemplary radicals include o-chlorophenyl, p-fluorophenyl andm-bromophenyl.

Hydroxy and mercapto radicals are also referred to herein as oxo andthioxo radicals, respectively, due to their tautomer formation.

Lower alkoxy radicals can be viewed as ethers formed from a 6-hydroxyisoxanthopterin and a before-described lower alkyl group. Exemplaryradicals include methoxy, ethoxy, propoxy, iso-propoxy, n-butoxy, andthe like. Phenyl-lower alkoxy radicals can similarly be viewed as ethersformed from a 6-hydroxy isoxanthopterin and a before-describedphenyl-lower alkyl radical. Exemplary of these materials are benzyloxy,2-phenylethoxy, 2-phenylpropoxy and the like.

Halo radicals preferably include chloro, bromo, as well as fluoro andiodo.

Lower alkylthio and phenyl-lower alkylthio radicals are sulfide ethersand are thus analogous to the oxygen ethers described above, as loweralkoxy and phenyl-lower alkoxy radicals, respectively.

A carboxy radical is a carboxylic acid (--CO₂ H) bonded to the6-position of the isoxanthopterin 8-aldoglycoside. A lower alkoxycarbonyl radical can be viewed as an ester of a 6-carboxyisoxanthopterin formed with a lower alkyl alcohol where the lower alkylportion of the alcohol is a lower alkyl radical as before-described.Exemplary esters are ethyl, methyl, t-butyl, neo-pentyl carboxylates,and the like. These esters can also be named ethoxycarbonyl,methoxycarbonyl, t-butoxycarbonyl and neo-pentoxycarbonyl, respectively.

Lower alkyl carboxy radicals are the before-described lower alkylradicals that further include a carboxy group. Lower alkoxy lower alkylcarbonyl radicals can be viewed as esters of substituent lower alkylcarboxy radicals with lower alkyl alcohols, which are describedimmediately above. Exemplary lower alkyl carboxy radicals includecarboxymethyl, 2-carboxyethyl, 2-carboxyhexyl and the like. Exemplarylower alkoxy lower alkyl carbonyl radicals include3-isopropoxycarbonylpropyl, 4-hexyloxycarbonylpentyl, and the like.

Carboxamido and lower alkylcarboxamido radicals can be viewed as beingformed from a carboxy or a lower alkyl carboxy substituent,respectively, and an amine. The carboxamido group has the formula CONR₃R₄ wherein R₃ and R₄ are the same or different and are selected from thegroup consisting of hydrogen and lower alkyl. Alternatively, NR₃ R₄together can form a heterocyclic ring having five or six atoms in thering. Exemplary useful amines include methylamine, propyl-amine,sec-butylamine, hexylamine, dimethylamine, methylethylamine,butylhexylamine, pyrrolidine, morpholine, piperidine, pyrrole and4-methylpiperazine. Unsubstituted carboxy amides (where R₃ and R₄ arehydrogen) are formed from ammonia as the amine.

Lower alkanoyl radical substituents, also known as lower acyl radicals,contain a carbonyl group bonded directly to the 6-position of theisoxanthopterin ring thereby making the compounds ketones, or analdehyde, as is appropriate. Exemplary lower alkanoyl groups includeformyl, acetyl, propionyl, 2-methylpropionyl, butyryl, 3-methylvaleryland the like. The acyl carbon of the radical is considered a part of the"lower" alkanoyl or acyl group.

Lower alkyloylthio or lower acylthio radicals can be viewed asthioesters formed from an appropriate 6-mercapto substituent of anisoxanthopterin derivative and a lower alkyl carboxylic acid. Exemplaryof such radicals are thioacetyl, thiopropionyl, thiohexanoyl and thelike.

A lower alkylene lower alkylcarboxylate radical can be viewed as anester of a substituent hydroxy lower alkyl radical and a lower alkylcarboxylic acid. Exemplary hydroxy lower alkyl substituents have beendiscussed previously, as have the lower alkanoyl (lower acyl) portionsof lower alkyl carboxylic acids that can be present in such esters.

The isoxanthopterin 8 aldoglycosides are weak bases, and as such canform acid addition salts. Pharmaceutically acceptable, non-toxic acidaddition salts of isoxanthopterin derivatives are useful herein, and canbe formed by treatment of the isoxanthopterin 8-aldoglycoside with anappropriate acid. Exemplary inorganic acids include hydrochloric,hydrobromic, sulfuric, phosphoric and the like acids. Exemplary orgainicacids include acetic, propionic, glycolic, pyruvic, malonic, succinic,maleic, fumaric, malic, tartaric, citric, benzoic, cinnamic, mendelic,methansulfonic, ethanesulfonic, benzenesulfonic, p-toluenesculfonic,salicyclic, p-aminosalicyclic and the like acids. Conversely, the acidaddition salt form can be converted to the free base form by treatmentwith alkali.

Useful isoxanthopterin derivatives also include 6-substituted carboxylicacids and lower alkyl substituted carboxylic acids, as already noted.Basic salts of those carboxylic acids are also contemplated, and areformed by treatment of the carboxylic acid with an appropriate alkalinereagent to form a 6-isoxanthopterin 8-aldoglycoside carboxylate cationsalt. Exemplary non-toxic cation salts of such carboxylic acids includesodium, potassium, zinc, aluminum, calcium, magnesium, and the like.

The 8-aldoglycoside portion (R₂) of the useful isoxanthopterinderivatives are cyclic, contain 5 or 6 carbon atoms, and are selectedfrom the group consisting of 1'-aldopentosidyl, 1'-aldohexosidyl,mono-deoxygenated 1'-aldopentosidyl, andmono-deoxygenated-1'-aldohexosidyl radicals.

Exemplary 1'-aldopentosidyl radicals are the 1'-radicals of ribose,arabinose, lyxose and xylose that are named 1'-ribofuranosidyl,1'-arabinofuranosidyl, 1'lyxofuranosidyl, and 1'xylofuranosidylradicals, respectively. Exemplary 1-aldohexosidyl radicals are the1'-radicals of glucose, galactose, mannose, gulose, allose, altrose, andrhamnose that are named 1'-glucopyranosidyl, 1'-galactopyranosidyl,1'-mannopyranosidyl 1'-gulopyranosidyl, 1'-allopyranosidyl,1'-altropyranosidyl, 1'-rhamnopyranosidyl, radicals, respectively. Anexemplary mono-deoxygenated 1'-aldopentosidyl radical is that ofdeoxyribose that is named the 1'(2'-deoxy)-ribofuranosidyl radical. Anexemplary mono-deoxygenated 1'-aldohexosidyl radical is that ofdeoxygulose, named the 1'-(2'-deoxy)gulopyranosidyl radical.

Useful aldoglycosidyl radicals can have one or more hydroxyl groupsesterified by a lower alkanoyl radical such as formyl, acetyl, propionylor hexanoyl, and also by a benzoyl radical. Aldoglycosidyl radicals arealso useful when etherified by lower alkyl, especially methyl and ethylradicals, while benzyl ethers are also useful.

Suitable aldoglycosidyl radicals conform to the formula ##STR4##

wherein n is one or zero;

R₅ is hydrogen, hydroxy, lower alkoxy such as methoxy and ethoxy (andothers as described before), benzyloxy, lower alkanoyloxy such asformyloxy, acetoxy (and other lower alkyl carboxylate radicals as aredescribed before) or benzoxy.

R₆ when present, as well as R₇ and R₈ are all the same. These radicalscan be hydroxy, a lower alkyl ether (lower alkoxy) such as methoxy andethoxy, a benzyl ether (benzyloxy), a lower alkanoyl radical (loweracyl) such as formyloxy, acetoxy, or a benzoate ester (benzoxy). When R₅is other than hydrogen, R₅ ═R₆ when present═R₇ ═R₈. Thus , anO-substituent, when present on one oxygen is present on all availablering substituent oxygens.

The bonds of the above formula are not intended to convey any particularstereo specific configuration, except at the 1'-position at which thebeta anomer is indicated.

In preferred practice, the aldoglycosidyl radical is selected from thegroup consisting of 1'-ribofuranosidyl, 1'-glucopyranosidyl, and1'-(2'-deoxy)ribofuranosidyl radicals. Thus, preferably, when n is zeroand R₅, R₇ and R₈ are all hydroxy, R₆ is absent, the aldoglycosidylradical is selected from the group consisting of 1'-ribofuranosidyl;when n is zero, R₅ is hydrogen and R₇ and R₈ are hydroxy, R₆ is absent,the aldoglycosidyl radical is 2'-deoxy-1'-ribofuranosidyl; and when n is1, and R₅ ═R₆ ═R₇ ═R₈ ═hydroxy, 1'-glucopyranosidyl is thealdoglycosidyl radical.

As already noted, the aldoglycoside is bonded from its 1'-position tothe 8-position of the isoxanthopterin derivative. When named as anisoxanthopterin derivative, that bonding can be described as an 8-1'bond. The beta anomer of the aldoglycoside is that preferred herein,although mixtures of alpha and beta anomers are also useful. Thealdoglycoside utilized is in the D stereo configuration and thatconfiguration is implied where it is not stated.

Structural formulas of exemplary isoxanthopterin derivatives useful inthe method of this invention are shown below, wherein R₁ and R₂ are asshown in the Table following the structural formula. ##STR5##

                  TABLE                                                           ______________________________________                                        R.sub.1        R.sub.2                                                        ______________________________________                                        methyl         1'-arabinofuranosidyl                                          iso-propyl     1'-lyxofuranosidyl                                              -n-butyl      1'-ribofuranosidyl                                              .sub.-t-butyl 1'-(2'-deoxy)ribofuranosidyl                                   neo-pentyl     1'-xylofuranosidyl                                              -n-hexyl      1'-gulopyranosidyl                                             benzyl         1'-galactopyranosidyl                                          phenethyl      1'-mannopyranosidyl                                            2-phenylpropyl 1'-(2',3',4',6'-tetra-O--                                                     acetyl)glucopyranosidyl                                        2-phenyl-3-    1'-(2',3',5'-tri-O--acetyl)-                                   methylpentyl   ribofuranosidyl                                                 -o-xylyl      1'-(2',3',5'-tri-O--acetyl)-                                                  arabinofuranosidyl                                              -p-(2-hexyl)phenyl                                                                          1'-(2'-deoxy-3',5'-di-O--                                                     methyl)ribofuranosidyl                                          .sub.--m-(iso-propyl)phenyl                                                                 1'-(2',3',4',6'-tetra-O--                                                     ethyl)glucopyranosidyl                                          -p-(trifluoromethyl)                                                                        1'-(2',3',5'-tri-O--benzyl)-                                   phenyl         ribofuranosidyl                                                 -o-methoxyphenyl                                                                            1'-(2',3',5'-tri-O--benzoyl)-                                                 ribofuranosidyl                                                 .sub.--m-sec-butoxyphenyl                                                                   1'-(2',3',4',6'-tetra-O--                                                     ethyl)glucopyranosidyl                                          -p-(2-ethylbutoxy)phenyl                                                                    1'-(2'-deoxy-3',5'-di-O--                                                     methyl)ribofuranosidyl                                          -o-chlorophenyl                                                                             1'-gulopyranosidyl                                              .sub.--m-bromophenyl                                                                        1'-allopyranosidyl                                              -p-fluorophenyl                                                                             1'-altropyranosidyl                                            hydroxy        1'-rhamnopyranosidyl                                           mercapto       1'-galactopyranosidyl                                          methoxy        1'-glucopyranosidyl                                            iso-propoxy    1'-xylofuranosidyl                                              -n-hexyloxy   1'-(2'-deoxy)ribofuranosidyl                                   benzoxy        1' -ribofuranosidyl                                            2-phenylethoxy 1'-lyxofuranosidyl                                             2-phenylpropoxy                                                                              1'-(2'-deoxy)gulopyranosidyl                                   chloro         1'-glucopyranosidyl                                            bromo          1'-(2'-deoxy)ribofuranosidyl                                   fluoro         1'-ribofuranosidyl                                             iodo           1'-ribofuranosidyl                                             ethylsulfido   1'-glucopyranosidyl                                            benzylsulfido  1'-arabinofuranosidyl                                          carboxy        1'-lyxofuranosidyl                                             carbomethoxy   1'-ribofuranosidyl                                             carbethoxy     1'-(2'-deoxy)ribofuranosidyl                                   carbo- .sub.-t-butoxy                                                                        1'-xylofuranosidyl                                             neo-pentoxycarbonyl                                                                          1'-glucopyranosidyl                                            2-carboxyethyl 1'-galactopyranosidyl                                          4-carboxybutyl 1'-mannopyranosidyl                                            ethyl carboxymethyl                                                                          1'-(2',3',4',6'-tetra-O--                                                     acetyl)glucopyranosidyl                                        sec-butyl carboxyethyl                                                                       1'-(2',3',5'-tri-O--acetyl)-                                                  ribofuranosidyl                                                sodium carboxy 1'-(2',3',5'-tri-O--acetyl)-                                                  arabinofuranosidyl                                             hydroxymethyl  1'-(2',3',5'-tri-O--methyl)-                                                  ribofuranosidyl                                                2-hydroxyethyl 1-(2',3',4',6'-tetra-O--                                                      benzyl)allopyranosidyl                                         1,2-dihydroxyethyl                                                                           1'(2'-deoxy)gulopyranosidyl                                    1,2,3-trihydroxypropyl                                                                       1'-rhamnopyranosidyl                                           ______________________________________                                    

Particularly preferred isoxanthopterin 8-aldoglycosides are those havinghydrogen, hydroxy, lower alkyl such as methyl, carboxy and lower alkylcarboxylate such as ethyl or methyl carboxylate (ethoxycarbonyl ormethoxycarbonyl) and polyhydroxy lower alkyl bonded at the 6-position,in which the 8-aldoglycoside portion of the molecule isbeta-1-ribofuranosidyl, beta-1'-(2'-deoxy)ribofuranosidyl, andbeta-1'-glucopyranosidyl. Exemplary of such particularly preferredmaterials are:

8-(1'-beta-D-ribofuranosidyl)isoxanthopterin;

8-(1'-beta-D-2'-deoxyribofuranosidyl)isoxanthopterin;

8-(1'-beta-D-glucopyranosidyl)isoxanthopterin;

6-hydroxy-8-(1'-beta-D-ribofuranosidyl)isoxanthopterin;

6-hydroxy-8-(1'-beta-D-2'-deoxyribofuranosidyl)-isoxanthopterin;6-hydroxy-8-(1'-beta-D-glucopyranosidyl)isoxanthopterin;6-methyl-8-(1'-beta-D-ribofuranosidyl)isoxanthopterin;6-methyl-8-(1'-beta-D-glucopyranosidyl)isoxanthopterin;

6-methyl-8-(1'-beta-D-2'-deoxyribofuranosidyl)isoxanthopterin;6-carboxy-8-(1'-beta-D-ribofuranosidyl)-isoxanthopterin;6-carboxy-8-(1'-beta-D-glucopyranosidyl)isoxanthopterin;6-carboxy-8-(1'-beta-D-2'-deoxyribofuranosidyl)isoxanthopterin;6-methoxycarbonyl-8-(1'-beta-D-ribofuranosidyl)isoxanthopterin;6-methoxycarbonyl-8-(1'-beta-D-2'-deoxyribofuranosidyl)isoxanthopterin;and

6-methoxycarbonyl-8-(1'-beta-D-glucopyranosidylisoxanthopterin.

Most preferred isoxanthopterin derivatives useful in the method of thisinvention are those compounds in which R₂ is the 1'-D-ribofuranosidylradical, and in which R₁ is selected from the group consisting ofhydrogen, methyl and carboxy. These compounds have structures thatconform to the formula ##STR6##

wherein R₁ is selected from the group consisting of hydrogen, methyl andcarboxy.

b. Contacting Compositions

The active ingredient isoxanthopterin derivative used in the presentinvention is contacted with animal cells whose responses are to bemodulated in vitro in cell culture or in vivo by administration to ananimal perorally or parenterally in customary dosage unit compositions,that is, as compositions in unit dosage form comprising aphysiologically tolerable carrier admixed with an effective dosage unitof the isoxanthopterin derivative.

The term "unit dosage" and its grammatical equivalents as used hereinrefer to physically discrete units suitable as unitary dosages for humanpatients and other warm blooded animals, each unit containing apredetermined effective amount of the active ingredient calculated toproduce the desired therapeutic effect in association with the requiredphysiologically tolerable carrier, e.g. a diluent of a vehicle. Thespecifications for the novel unit dosage forms of this invention aredictated by and are directly dependent on (a) the unique characteristicsof the active isoxanthopterin derivative ingredient and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active ingredient for therapeutic use invitro, as well as in vivo in humans and other animals.

Examples of suitable unit dosage forms in accord with this invention aretablets, capsules, pills, powder packets, granules, wafers, and thelike, segregated multiples of any of the foregoing, as well as liquidsolutions, emulsions and suspensions. Liquid compositions can beadministered in usual manners such as subcutaneously, intraperitoneally,intermuscularly, perorally or the like.

The amount of active ingredient that is administered in vivo depends onthe age and weight of the patient, the particular condition to betreated, the frequency of administration, and the route ofadministration. The dose range can be about 0.01 to about 200 milligramsper kilogram of body weight, more preferably about 0.1 to about 25milligrams per kilogram of body weight, and most preferably about 1 toabout 10 milligrams per kilogram of body weight. The human adult dose isin the range of about 5 to about 1400 milligrams daily, given either asa single dose or in 3 or 4 divided doses. Veterinary dosages correspondto human dosages with the amounts administered being in proportion tothe weight and metabolic rate of the animal as compared to adult humans.

Concentrations for the in vitro contacting of animal cells are about1×10⁻⁸ molar to about 1×10⁻³ molar for cell concentrations of about2-5×10⁶ cells per milliliter. More preferably, the concentration isabout 1×10⁻⁷ molar to about 1×10⁻⁴ molar, and still more preferablyabout 3×10⁻⁶ molar to about 3×10⁻⁵ molar at the same cellconcentrations. A composition for contacting the animal cells can be asolid or a liquid. The isoxanthopterin derivative can be admixed as asuspension of solid isoxanthopterin derivative in a solid or liquidphysiologically tolerable carrier, or dissolved as a solute or suspendedin the carrier, or a combination thereof.

Physiologically tolerable carriers are well known in the art. Exemplaryof liquid carriers are sterile aqueous solutions that can contain nomaterials in addition to the active ingredient and water, or contain abuffer such as sodium phosphate at physiological pH value, physiologicalsaline or both, such as phosphate-buffered saline. Still further,aqueous carriers can contain more than one buffer salt, as well as saltssuch as sodium and potassium chlorides, dextrose and other solutes.These latter carries are exemplified by Ringer's injection, dextroseinjection, dextrose and sodium chloride injection and lactated Ringer'sinjection.

Liquid compositions can also contain liquid phases in addition to and tothe exclusion of water. Exemplary of such additional liquid phases areglycerin, vegetable oils such as cottonseed oil or sesame oil, andwater-oil emulsions.

Exemplary solid carriers include those materials usually used in themanufacture of pills or tablets, and include corn starch, lactose,dicalcium phosphate, thickeners such as tragacanth and methylcelluloseU.S.P., finely divided SiO₂, polyvinylpyrrolidone, magnesium stearateand the like. Additionally, the solid carrier can include biodegradableand nonbiodegradable polymers, polypeptide carriers, affinity carrierssuch as AFFI-GEL 601 (phenyl boronate resin available from Bio-RadLaboratories, Richmond CA), and liposomes as are known in the art.Antioxidants such as methylparaben and proplyparaben can be present inboth solid and liquid compositions, as can sweeteners such a cane orbeet sugar, sodium saccharin, sodium cyclamate and the dipeptide methylester sweeteneer sold under the trademark NUTRASWEET (aspartame) by G.D. Searle Co.

Contact between the composition and animal cells is maintained for atime period sufficient for the contacted cells manifest the modulationof their cellular response. Modulation of cellular response (activity)can itself be manifest in enhanced antibody secretion, enhanced T helperactivity, enhanced cytokine production, and the like.

For use in vivo, contact between animal cells and optimal concentrationsof the composition is typically maintained for a time period sufficientfor the animal to clear the isoxanthopterin derivative from its body asby metabolism, excretion or both processes. That time period can belonger than that required for a cellular response to be manifest.Contact with an individual unit dose is thus typically maintained for atime period of about one day to about seven days. Continual contact canbe advantageous for an immunodeficient animal host.

Contact in vitro can be maintained for a period of time sufficient forone of the before-described cellular functions to become manifest asdetermined by standard assay techniques. Such maintenance timestypically take about one to about seven days of time, and more usuallyabout 2 to about 4 days.

C. Modulated Cellular Responses

1. In vitro adjuvanticity

Contacting animal antibody-producing cells with a composition usefulherein provides an adjuvant effect on the primary antibody response toSRBC and other immunogens (antigens) when evaluated in vitro. The immuneresponse-modulating composition and effective amount of immunogen (sheepred blood cells; SRBC) are typically admixed to contact the cellssubstantially simultaneously. The words "antigen" and "immunogen" areused interchangeably.

At optimal concentration, a composition containing an effective amountof a useful isoxanthopterin derivative enhances the response to SRBC byabout 2-to 6-fold. The effect is dose dependent. Enhancement of theantibody response cannot be accounted for by the additive effects of thespecific response to SRBC and the polyclonal response to theisoxanthopterin derivative.

The adjuvant effect of compositions containing a useful isoxanthopterinderivative is exerted on immunogen-experienced (primed) as well on naivecells. Both responses are enhanced by contacting the cells withcompositions containing an effective amount of isoxanthopterinderivative. This adjuvant effect is dependent upon the concentration ofimmunogen added to culture.

While immune responses; i.e., responses of B lymphocytes or B cells, areobserved to be enhanced at all immunologically effective doses ofimmunogen, the degree of enhancement is usually greatest at optimal ornear optimal immunogen concentrations. Additionally, adjuvanticity ofisoxanthopterin derivatives is synergistic with immunogen and not justdue to the sum of independent immunogen-specific and polyclonal(nonspecific) responses.

Enhancement of antibody produced by compositions containing anisoxanthopterin derivative involves not only naive,immunogen-inexperienced B cells, but also immunogen-experienced ormemory B cells, as already noted. Thus, the primary IgM as well as thesecondary IgM and IgG responses to immunogen (antigen) are augmented bycontacting B cells with a composition containing an effective amount ofan isoxanthopterin derivative as active ingredient, and maintaining thatcontact as discussed herein.

For memory responses, B cells are primed by treatment with an effective,priming, amount of an immunogen, as is well known. That primingtreatment can be in the presence or absence of an immuneresponse-modulating composition. When contacted in the presence of sucha composition, treatment of the B cells with a priming amount ofimmunogen is preferably substantially simultaneous; i.e., within about12 hours, with contacting of the cells with a composition useful in thisinvention. More preferably, the immunogen is included in the immuneresponse-modulating composition, unless its effect is impaired by beingin that composition, as by denaturation.

A modulated cellular response can thus be obtained by contacting B cellssubstantially simultaneously with an effective, priming amount ofimmunogen and an immune response-modulating composition useful herein,followed, after a primary immune response is obtained, by an additionalcontacting of the primed cells with a further effective amount ofimmunogen (antigen) alone or substantially simultaneously with a furtheramount of immune response-modulating composition.

When the B cells are primed in the absence of a composition usefulherein, adjuvanticity can be demonstrated when the primed cells areagain treated with a further effective amount of immunogen substantiallysimultaneously with those primed cells being contacted with acomposition useful herein. A modulated cellular response can thus bemanifest by treating B cells that are primed to an effective, primingamount of immunogen with a further, effective amount of immunogen and animmune response-modulating composition useful herein that is contactedwith the B cells at preferably substantially the same time (within about12 hours) as those cells are treated with the second, effective amountof immunogen.

Isoxanthopterin derivative-containing compositions useful herein arethought to enhance the primary humoral immune response by actingdirectly upon the B cell and/or the immunogen-presenting cell. Thus, useof these derivatives enhances the antibody response mounted againstT-independent antigens; i.e., responses that involve B cells andimmunogen-presenting cells. In addition, compositions containing anisoxanthopterin derivative can replace the need of B cells for T helpercells, as discussed hereinafter, and therefore exert their adjuvanteffect in cultures initiated in the absence of intact, functional Tcells. A replacement of T cells with T cell helper activity contained inmixed lymphocyte culture (MLC) supernates does not diminish the abilityof an isoxanthopterin derivative to augment the antibody response.

Still further, the synergy observed between the soluble T cell signalcontained in MLC supernate and the isoxanthopterin derivative-containingcomposition indicates that the signal supplied by each is qualitativelydistinct. This synergy is observed over a range of supernateconcentrations, indicating that the isoxanthopterin derivative is notsimply providing more of the same "signal" that T cells provide. Acomparable degree of synergy can be observed when such B cell culturesare supplemented with T cells rather than with T cell-like supernates(which are in fact T cell derived), and are contacted in the presence ofimmunogen with an isoxanthopterin derivative-containing compositionuseful in this invention.

T cell-mediated effects of the adjuvanticity of isoxanthopterinderivatives are not ruled out by the observation of T-independence forthat adjuvanticity; i.e., the existance of a T cell-independent factdoes not bear upon the existance of a T cell-dependent phase. Thus, moresubstantial enhancement can be observed from a composition containingthe isoxanthopterin derivative under conditions of stimulation with lowdoses of T-dependent and T-independent type 2 antigens (T cell dependentsituations) than with T-independent type 1 antigens (more completely Tcell-independent), which suggests the presence of a T cell-dependentcomponent. Moreover, isoxanthopterin derivatives are thought to act(either directly or indirectly) on precursors of T helper cells toincrease the ability of a population of such cells to support anantibody response to immunogen.

2. In vivo modulation of immune response

Immunopotentiating effects on the primary antibody (B cell) response toSRBC in vivo are observed when a liquid composition containing anisoxanthopterin derivative useful herein is contacted with animal cellsas by injecting the composition into CBA/CaJ mice thirty minutes afterinjection of the SRBC immunogen; i.e., substantially simultaneously.Relatively high dosages, e.g., about 2.5 milligrams per animal (aboutone-tenth gram per kilogram), are tolerated by the animals.

Immunogen dose dependency in the above mice to adjuvant effects of aconstant level of the isoxanthopterin derivative injectedintraperitoneally (i.p.) are compared with normal saline (NS) i.p.injections as a control. While there is an enhancement in the immuneresponse at all useful (effective) levels of immunogen injection,typically, the enhancement becomes greater as the magnitude of theunderlying response increases.

In vivo modulation of animal cellular responses as in theabove-described primary immunization can also be effected as describedbefore in relation to in vitro modulation of secondary immune responsesof B cells.

3. T cell-replacing activity

A method of this invention can be used to substitute for T cells in theantibody response to a T-dependent immunogen. Here, T cells are depletedin vitro by treatment with complement and monoclonal anti-thy 1.2antibodies and are cultured with or without SRBC as immunogen in thepresence of compositions containing incremental concentrations of anisoxanthopterin derivative. Under these conditions, isolated B cellcultures are unable to respond to immunogen unless supplemented with a Tcell-like signal, such as is contained by a composition containing aneffective amount of an isoxanthopterin derivative. The modulatedcellular response is dose-dependent as well as immunogen-dependent. Inaddition, this response cannot be attributed to nonspecific polyclonalactivation of B cells.

Use of a method of this invention can provide a T cell-like signal toimmunogen-stimulated B cells, supplanting the need for T cellsaltogether under conditions of an otherwise T-dependent response. Thus,supplementation of murine B cell cultures, depleted of thy 1.2-bearing Tcells, with a composition containing an effective amount of anisoxanthopterin derivative replaces the requirement for T helper cellsin the generation of a primary antibody response to SRBC. This occurswhether splenocytes are depleted of T cells by in vitro treatment withmonoclonal anti-thy 1.2 antibodies and complement, or by in vivoinjection of rabbit anti-mouse thymocyte serum (ATS) followed by invitro treatment with ATS, anti-thy 1.2, anti-Lyt 1, and anti-Lyt 2 andcomplement as described by Harwell et al., J. Exp. Med. 152:893 (1980).

The mechanism of action of a composition of this invention is thought tobe distinct from that of T cell-derived lymphokines and the Tcell-replacing (or B cell stimulating) activity contained therein. Thisis shown by the synergistic effects of an isoxanthopterin derivative andT helper factor generated in MLC supernates wherein the anti-SRBC plaqueforming cells (PFC) response supported by the supernates is amplified byaddition of compositions containing an effective amount of anisoxanthopterin derivative.

4. Adjuvanticity by oral administration

Adjuvanticity of the compositions of this invention administered by invivo i.p. or subcutaneous injection was discussed hereinbefore.Adjuvanticity of the compositions of this invention that areadministered orally through a tube extending into the stomachs of theanimals or by use of a round-tipped feeding needle extending into theesophagus of each animal can also be shown.

Here, SRBC are injected i.p. and the PFC determinations are made sevendays after the initial i.p. injection of the SRBC. The compositionsuseful herein containing an effective amount of isoxanthoperinderivative are administered perorally within either the same 24-hourtime period as the immunogenic dose of SRBC or 72 hours thereafter.Administration of a composition of this invention to contact the animalcells provides an enhanced primary response to the immunogen whethercontacted with the animal cells within the same 24-hour period thatthose cells were subjected to the immunogen, or 72 hours thereafter.

For in vitro contacting, the cells are typically cultured in a mediumthat contains the isoxanthopterin derivative at a before-describedconcentration. For in vivo contacting, the composition is administeredto the animal one or more times and is maintained in the animal untilthe last-administered dose is cleared from the animal's body, andthereby from contact with the animal's cells, by natural bodilyprocesses as discussed before.

D. Best Mode For Carrying Out The Invention Example 1.

Tablets

Tablets are compounded from the following ingredients:

    ______________________________________                                                         Parts by Weight                                              ______________________________________                                        8-(1'-Beta-D-ribofuranosidyl)                                                                    0.5                                                        isoxanthopterin                                                               Lactose, powdered  37.4                                                       Corn starch, dry   35.5                                                       Finely divided SiO.sub.2                                                                         5.6                                                        Polyvinylpyrrolidone                                                                             0.6                                                        Magnesium stearate 0.4                                                                           80.0                                                       ______________________________________                                    

The isoxanthopterin derivative is thoroughly admixed with the lactose,25.0 parts by weight of the corn starch, and 4.0 parts by weight of theSiO₂. The resulting admixture is then uniformly moistened with a 5%ethanolic solution of polyvinylpyrrolidone. The moist mass is thenpassed through a one-millimeter mesh screen to produce a granulate. Theproduced granulate is dried for about 24 hours at 60° C. in a dryingchamber. The dried granulate is again passed through a one-millimetermesh screen. 70.0 Parts of the obtained granulate are admixed in asuitable mixer with a mixture consisting of the remainder of the SiO₂,the remainder of the corn starch and all of the magnesium stearate,which mixture previously had been passed through a one-millimeter meshscreen. The thus-obtained admixture is then pressed into tabletsweighing 800 milligrams each and containing 5 milligrams of theisoxanthopterin.

Example 2

Starch Capsules

Capsule contents are compounded from the following ingredients:

    ______________________________________                                                        Parts by Weight                                               ______________________________________                                        6-Carboxy-8-(1'-beta-D-                                                                         1.0                                                         glucopyranosidyl)-                                                            isoxanthopterin                                                               Lactose           450.0                                                       Corn Starch       549.0                                                                         1000.0                                                      ______________________________________                                    

The isoxanthopterin derivative is gradually admixed with the lactose.When all of the lactose has been admixed, the obtained admixture isblended with the corn starch. The resulting blend is then filled intocapsules holding 1.0 gram of the blend. Each capsule contains 1.0milligram of the isoxanthoperin derivative.

Example 3

Tablets

A lot of 10,000 tablets, each containing 50 milligrams of6-methyl-8-(1'-beta-D-deoxyribofuranosidyl)-isoxanthopterin, is preparedfrom the following types and amounts of ingredients:

    ______________________________________                                        6-Methyl-8-(1'-beta-D-   500 grams                                            deoxyribofuranosidyl)isoxanthopterin                                          Dicalcium Phosphate      1000 grams                                           Methyl cellulose, U.S.P. (15 cps)                                                                      75 grams                                             Talc                     150 grams                                            Corn Starch              250 grams                                            Magnesium stearate       25 grams                                                                      2000 grams                                           ______________________________________                                    

The isoxanthopterin derivative and dicalcium phosphate are mixed well,granulated with 7.5 percent solution of methyl cellulose in water,passed through a No. 8 screen (U.S. Standard Sieve Series) and driedcarefully. The dried granules are passed through a No. 12 screen (U.S.Std. Sieve Series), mixed thoroughly with the talc, starch and magnesiumstearate, and compressed into tablets.

These tablets are useful for enhancing antibody production whenadministered perorally at a dose of one to three tablets about every sixto eight hours.

Example 4

Injectable Preparation

A sterile preparation suitable for subcutaneous or intracavitaryinjection and containing 50 milligrams of6-carboxyethyl-8-(1'-beta-D-ribofuranosidyl)isoxanthopterin in eachmilliliter of ingredients is prepared from the following types andamounts of ingredients:

    ______________________________________                                        6-Carboxyethyl-8-(1-beta-D-                                                                          5     grams                                            ribofuranosidyl)isoxanthopterin                                               Physiological saline   98    milliliters                                      Cottonseed oil (or sesame oil)                                                                       2     milliliters                                      ______________________________________                                    

The isoxanthopterin derivative and saline are admixed and sonicated fora period of time sufficient to provide a substantially homogenousdispersion. The cottonseed oil (or sesame oil) is thereafter admixed andthe new admixture is similarly homogenized to provide an emulsion. Afteremulsification, one to three percent of the final volume of this sterilepreparation are injected subcutaneously or intraperitoneally once a weekto enhance humoral immunity.

Example 5

Aqueous Preparation for Oral Use

An aqueous preparation for oral use containing in each 5 milliliters (1teaspoon) 5 milligrams of6-carboxy-8-(1'-beta-D-glucopyranosidyl)isoxanthopterin is prepared fromthe following ingredients:

    ______________________________________                                        6-Carboxy-8-(1'-beta-D-                                                                              1.0    grams                                           glucopyranosidyl)-isoxanthopterin                                             Methylparaben, U.S.P.  0.75   grams                                           Propylparaben U.S.P.   0.25   grams                                           Saccharin sodium       1.25   grams                                           Cyclamate sodium       0.25   grams                                           Glycerin               300    milliliters                                     Tragacanth powder      1.0    grams                                           Orange oil flavor      1.0    grams                                           F.D. and C. orange dye 0.75   grams                                           Deionized water, q.s. to                                                                             1000   milliliters                                     ______________________________________                                    

A dose of one teaspoon two to four times per day is useful for enhancinghumoral immunity.

Cell Contacting Conditions

Lymphocyte cultures

The serum-containing culture medium is prepared to contain the followingper 100 millimeters: 91.9 milliliters RPMI 1640 (Flow Laboratories,Inc., Rockville, MD), 0.1 milliliters of 100×glutamine, 1.0 milliliterof 100×sodium pyruvate, 1.0 milliliter of 50×nonessential amino acids,1.0 milliliter of water contain 10⁴ units of penicillin G and 10⁴micrograms of streptomycin, and 5.0 milliliters of a supportive lot offetal calf serum (FCS). These ingredients are admixed to apparenthomogeneity. Spleen cell suspensions and populations enriched forsplenic B cells are prepared as described in Goodman et al., J.Immunol., 121: 1905 (1978).

For evaluation of the primary humoral immune response to sheeperythrocytes (SRBC), 5×10⁶ to 10⁷ murine spleen cells are cultured in1.0 milliliter of 5% FCS-containing medium for 4 or 5 days in thepresence of immunogen. Cells are incubated in culture trays (3424,Costar, Cambridge, MA) at 37° C. in a humidified atmosphere of 10% CO₂in air using tissue culture boxes (CBS Scientific, Del Mar, CA) that arerocked at a frequency of 7 cycles per minute. Pooled SRBC are availablefrom the Colorado Serum Co., Denver CO.

Assay of plaque forming cells (PFC)

PFC secreting antibodies against SRBC are evaluated after 4 or 5 days ofculture using a modification of the hemolytic plaque assay of Jerne andNordin, Science, 140:405 (1963).

Mice

CBA/CaJ mice, 8-16 weeks of age, are purchased from the JacksonLaboratory, Bar Harbor, ME. All mice are maintained on Wayne Lab Blox F6pellets (Allied Mills, Inc., Chicago, IL) and chlorinated wateracidified with HCl to a pH value of 3.0.

Cell preparations

Spleen and thymus cell suspensions are prepared as described in Goodmanet al., J. Immunol., 121:1905 (1978). Spleen cells enriched for Tlymphocytes are prepared by passage through nylon wool (NW) columnsaccording to the protocol of Julius et al., Eur. J. Immunol., 3:645(1973). B cell-enriched populations are prepared by treating 10⁸ spleencells with a 1:1000 dilution of monoclonal anti-Thy 1.2 antibody (NewEngland Nuclear, Boston, MA) for 30 minutes at 4° C. Treated cells arecentrifuged at 280×gravity for 10 minutes, antibodies are removed, andthe cells are resuspended in a 1:6 dilution of CBA RBC-absorbed guineapig complement at 37° C. for 45 minutes. Cells are then washed andcultured as described before.

Materials

Isoxanthopterin-8-beta-D-ribonucleoside was a gift of Dr. WolfgangPfleiderer, University of Konstanz, Konstanz West Germany.8-Oxoguanosine (8-oxoGuo) was a gift from Dr. Roland Robins, ICNPharmaceuticals, Costa Mesa, CA. Human IL-2, lot 1464-52, was obtainedas a partially purified preparation from Electro-Nucleonics, Inc.,Silver Springs MD. This preparation was found to be free ofinterferon-gamma activity.

Injections

Mice were injected i.p. with a suspension of washed SRBC at differentconcentrations in saline. At various times thereafter, different amountsof isoxanthopterin derivatives are injected i.p. or subcutaneously. Theisoxanthopterin derivative is typically injected as a suspension insaline, in a water-oil emulsion, or in 10 mg/ml of sodium carboxymethylcellulose (CMC) in normal saline (NS) or physiologic saline. For oralfeeding studies, mice are intubated with poly(propylene) cathetersextending from the mouth to the stomach, or with a round-tipped 20 gaugefeeding needle as discussed before, and the measured amounts ofcompositions are introduced therethrough.

Example 6

Enhancement of Primary Antibody Response

5×10⁶ Viable CBA/CaJ mouse spleen cells were cultured in 1 ml ofserum-containing medium in the presence or absence of SRBC withincremental amounts of 8-(1'-beta-D-ribofuranosidyl)isoxanthopterinpresent over a concentration range of zero (None) through 1×10⁻⁴ molar.PFC to SRBC were counted after 4 days of culture.

The results of two studies using the murine system as describedhereinbefore are shown in Table 1, below.

                  TABLE 1                                                         ______________________________________                                        Adjuvanticity of Isoxanthopterin-8-beta-D                                     Riboside on the Murine                                                        Primary Antibody Response                                                                       Direct Anti-SRBC                                                              PFC/Culture                                                 Antigen.sup.1                                                                            Nucleoside.sup.2                                                                           Study 1  Study 2                                      ______________________________________                                        None       None         62        35                                          SRBC       None         468      495                                                     Isox. Rib. at                                                      SRBC       10.sup.-8    638      418                                          SRBC       10.sup.-7    773      948                                          SRBC       10.sup.-6    778      660                                          SRBC       10.sup.-5    490      540                                          SRBC       10.sup.-4    208      178                                                     8-oxoGuo at:                                                       SRBC       3 × 10.sup.-4                                                                        6217     --                                           ______________________________________                                         .sup.1 5 × 10.sup.6 SRBC used as antigen per culture.                   .sup.2 Nucleosides are listed as being absent (None) or at a concentratio     in moles per liter, e.g., 10.sup.-5 Isox. Rib. = isoanthropterin riboside     8oxoGuo = 8oxoguanosine.                                                 

As can be seen, a dose-dependent enhancement of PFC in the presence ofSRBC and isoxanthopterin derivative over those of the culturescontaining the SRBC alone are noted. The activity of this compound at aconcentration as low as 10⁸ molar was particularly surprising.

Example 7

Enhancement of Secondary Antibody Response

10⁷ Viable SRBC-primed CBA/CaJ mouse spleen cells are cultured inserum-containing medium in the presence or absence of variableconcentrations of 8-(1'-beta-D-ribofuranosidyl)isoxanthopterin with6×10⁵ pooled SRBC per ml of culture. Direct PFC to SRBC are determinedafter 4 days of culture. Similar cultures are prepared as controls usingthe same incremental amounts of SRBC, but lacking the isoxanthopterinderivative.

PFC are increased in the cultures containing both the isoxanthopterinand SRBC as compared to cultures containing SRBC above.

Use of 6-methyl-8-(1'-beta-D-ribofuranosidyl)isoxanthopterin and6-carboxy-8-(1'-beta-D-ribofuranosidyl)isoxanthopterin provides similarresults.

Example 8

In Vivo Modulation of Immune Response

CBA/CaJ mice are injected with 6×10⁶ SRBC intraperitoneally, and threedays later with incremental amounts of6-methyl-8-(1'-beta-D-ribofuranosidyl) isoxanthopterin in CMC.Concentrations of zero through 2.5 milligrams per animal are used.Assessment of direct PFC to SRBC 5 days thereafter shows adose-dependent enhancement of the antibody response.

Similar results are obtained using8-(1'-beta-D-ribofuranosidyl)isoxanthopterin and6-carboxy-8-(1-beta-D-ribofuranosidyl)isoxanthopterin as theisoxanthopterin derivative.

Example 9

In Vitro Enhancement of Human Primary Immune Response

Human peripheral blood lymphocytes (PBL) were prepared from normalheparinized venous blood by Ficoll-diatrizoate density gradientcentrifugation. PBL were depleted of suppressor T cells bearing thehistamine type 2 receptor by adhering them to the surfaces ofhistamine-rabbit albumin-coated plastic petri dishes (Cell-ect No. 2kit; Seragen, Boston, MA) and by recovering the nonadherent cells bypanning as described by Wysocki and Sato, Proc. Natl. Acad. Sci. USA,75:2844 (1978) and modified by Cavagnaro and Osband, Biotechniques,January/February:30 (1983).

The tissue culture medium employed in these studies was prepared asfollows: One hundred milliliters (ml) contained 87.9 ml RPMI 1640 (FlowLaboratories, Rockville, MD), 0.1 ml 100×glutamine, 1.0 ml of 1.0 MHEPES buffer (Microbilogical Associates, Betheseda, MD), 1.0 ml of watercontaining 10⁴ U of penicillin G and 10⁴ micrograms of streptomycin, and10 ml of fresh autologous heat-inactivated plasma. For evaluation of theprimary humoral immune response to SRBC, lymphoid cells were cultured ata density of 2×10⁶ /ml in a volume of 1.0 ml containing 5×10⁶ SRBC asantigen (Colorado Serum Co., Denver, CO) together with IL-2 and theisoxanthopterin ribonucleoside.

Enumeration of plaque-forming cells (PFC) that secrete antibodiesagainst SRBC was accomplished after 6 days of culture by using amodification of the hemolytic plaque assay of Jerne and Nordin, Science140:405 (1963). The cells were brought up in complete medium beforeplaquing; they were plaqued in standard low M_(r) agarose (Bio-RadLaboratories, Richmond, CA), and were incubated in SRBC-absorbed guineapig complement for 1 hour after a 1.5-hour incubation withoutcomplement.

The results of two studies are shown in Table 2, below.

                  TABLE 2                                                         ______________________________________                                        Adjuvanticity of Isoxanthopterin-8-beta-D-                                    Ribonucleoside on the Human                                                   Primary Antibody Response                                                                     Direct Anti-SRBC                                                              PFC/Culture.sup.3                                             Antigen.sup.1                                                                           Nucleoside.sup.2                                                                          Study 1    Study 2                                      ______________________________________                                        None      None        7           12                                          SRBC      None        5,383      363                                                    Isox. Rib. at                                                       SRBC      10.sup.-8   5,258      --                                           SRBC      10.sup.-7   6,017      --                                           SRBC      10.sup.-6   5,483      --                                           SRBC      10.sup.-5   7,592      665                                          SRBC      3 × 10.sup.-5                                                                       --         248                                          SRBC      10.sup.-4   32,000     180                                          SRBC      3 × 10.sup.-4                                                                       --         230                                                    8-oxoGuo at:                                                        SRBC      3 × 10.sup.-4                                                                       --         1175                                         SRBC      10.sup.-3   10,100     --                                           ______________________________________                                         .sup.1 SRBC used as antigen at 5 × 10.sup.6 cells per culture.          .sup.2 Nucleosides are listed as being absent (None) or at a concentratio     in moles per liter, e.g., 10.sup.-5 Isox. Rib. = isoxanthopterin riboside     8oxoGuo = 8oxoguanosine.                                                      .sup.3 Direct antiSRBC plaqueforming cells (PFC) per culture were             determined as described before using PBL from two different human donors.

As can be seen from the above data, a dose-dependent antigen-specificenhancement was obtained with isoxanthopterin ribonucleoside. Theresults obtained were found at a higher concentration of theisoxanthopterin derivative than was optimal in the mouse system ofExample 6. These results are consistent with results in murine and humansystems observed using 8-substituted guanosines. See, Goodman andHennen, Cell, Immunol., 102:395 (1986) and Goodman and Weigle, J.Immunol., 135:3284 (1985).

Example 10

T Cell Replacing Activity

4×10⁶ Viable CBA/CaJ mouse splenic cells are treated first withcomplement-fixing monoclonal antibodies that immunoreact with thy 1.2antigens of T cells and second with complement to lyse any T cellspresent (New England Nuclear, Boston, MA). The cells so treated arethereafter grown with or without SRBC as immunogen in serum-containingmedia further containing incremental amounts of8-(1'-beta-D-ribofuranosidyl)isoxanthopterin ranging in amount from zerothrough 10⁻⁴ molar. Direct PFC to SRBC are determined 4 days thereafter.The results of this study indicate that the presence of theisoxanthopterin derivative assists in inducing a B cell response to theimmunogen, and the result induced is dose-dependent. Thus, contactingthe unlysed splenic cells (i.e., B cells) with a composition usefulherein provides a T cell-like "signal" to those unlysed cells.

Similar results are obtained using6-methyl-8-(1'-beta-D-ribofuranosidyl)isoxanthopterin and6-carboxy-8-(1'-beta-D-ribofuranosidyl)isoxanthopterin to replace theabove isoxanthopterin derivative.

The present invention has been described with respect to preferredembodiments. It will be clear to those skilled in the art thatmodifications and/or variations of the disclosed subject matter can bemade without departing from the scope of the invention set forth herein.

What is claimed is:
 1. A method of modulating an animal immune response comprising contacting animal cells with a composition containing a diluent amount of a physiologically tolerable carrier admixed with an immune response-modulating effective amount of an active ingredient that is an isoxanthopterin derivative whose structure conforms to that of the formula ##STR7## wherein R₁ is a radical selected from the group consisting of hydrogen, lower alkyl, hydroxy lower alkyl, polyhydroxy lower alkyl, phenyl, phenyl-lower alkyl, lower alkyl phenyl, lower alkoxy phenyl, halophenyl, trifluoromethyl phenyl, hydroxy, oxo, lower alkoxy, phenyl-lower alkoxy, halo, mercapto, thioxo, lower alkylthio, lower alkyloylthio, phenyl-lower alkylthio, lower alkanoyl, carboxy, lower alkoxy carbonyl, lower alkylcarboxy, lower alkylene lower alkylcarboxylate, lower alkoxy lower alkyl carbonyl, and carboxamido and lower alkyl carboxamido in which the carboxamido group has the formula CONR₃ R₄ wherein R₃ and R₄ are the same or different and are selected from the group consisting of hydrogen and lower alkyl or NR₃ R₄ together form a heterocyclic ring having five or six atoms in the ring;R₂ is a beta-bonded aldoglycoside radical selected from the group consisting of 1'-aldopentosidyl, 1'-aldonexosidyl, mono-deoxygenated 1'-aldopentosidyl, and mono-deoxygenated 1'-aldohexosidyl and their O-substituted lower alkyl, lower alkanoyl, benzyl and benzoyl derivatives wherein an O-substituent, if present on one oxygen, is present on all available ring substituent oxygens; the tautomers of said isoxanthopterin derivative; and the pharmaceutically acceptable salts of said isoxanthopterin derivative; and maintaining said contact for a time period sufficient for said cells to modulate their immune response.
 2. The method in accordance with claim 1 wherein R₂ is selected from the group consisting of 1'-ribofuranosidyl, 1'-(2'-deoxy)ribofuranosidyl and 1-glucopyranosidyl radicals.
 3. The method in accordance with claim 2 wherein the structure of said isoxanthopterin compound conforms to the formula ##STR8##
 4. The method in accordance with claim 3 wherein R₁ is selected from the group consisting of hydrogen, methyl, and carboxy.
 5. The method in accordance with claim 1 wherein the structure of said aldoglycosidyl radical conforms to the formula ##STR9## wherein n is zero or more; R₅ is selected from the group consisting of hydrogen, hydroxy, lower alkoxy, benzyloxy, lower alkanoyloxy, and benzoxy;R₆, R₇ and R₈ are the same and are selected from the group consisting of hydroxy, lower alkoxy, benzyloxy, lower alkanoyloxy, and benzoxy; and where R₅ ═R₆ ═R₇ ═R₈ when R₅ is other than hydrogen.
 6. The method in accordance with claim 5 wherein n is zero, and R₅ R₆, R₇ and R₈ are hydroxy.
 7. The method in accordance with claim 5 wherein n is 1, and R₅, R₆, R₇ and R₈ are hydroxy.
 8. The method in accordance with claim 1 wherein said cells are leukocytes.
 9. The method in accordance with claim 8 wherein said leukocytes are B lymphocytes.
 10. The method in accordance with claim 9 including the additional step of treating said B lymphocytes with an effective amount of an immunogen prior to contact with said composition, said immunogen treatment priming said B-lymphocytes.
 11. The method in accordance with claim 10 wherein said B lymphocytes are contacted with said composition in conjunction with an additional amount of an immunogen previously used to prime said B lymphocytes for an immune response.
 12. The method in accordance with claim 9 wherein said B lymphocytes are contacted with said composition substantially simultaneously with an effective amount of an immunogen.
 13. The method in accordance with claim 1 wherein the animal cells contacted with said composition are leucocytes.
 14. The method in accordance with claim 13 wherein said animal cells are contacted in vitro.
 15. A method of enhancing cellular immune responses of leucocytes comprising the steps of:contacting leucocytes with a composition containing a diluent amount of a physiologically tolerable carrier admixed with an immune response-modulating effective amount of an active ingredient that is an isoxanthopterin derivative whose structure conforms to that of the formula ##STR10## wherein R₁ is a radical selected from the group consisting of hydrogen, lower alkyl, hydroxy lower alkyl, polyhydroxy lower alkyl, phenyl, phenyl-lower alkyl, lower alkyl phenyl, lower alkoxy phenyl, halophenyl, trifluoromethyl phenyl, hydroxy, oxo, lower alkoxy, phenyl-lower alkoxy, halo, mercapto, thioxo, lower alkylthio, lower alkyloylthio, phenyl-lower alkylthio, lower alkanoyl, carboxy, lower alkoxy carbonyl, lower alkylcarboxy, lower alkylene lower alkylcarboxylate, lower alkoxy lower alkyl carbonyl, and carboxamido and lower alkyl carboxamido in which the carboxamido group has the formula CONR₃ R₄ wherein R₃ and R₄ are the same or different and are selected from the group consisting of hydrogen and lower alkyl or NR₃ R₄ together form a heterocyclic ring having five or six atoms in the ring; R₂ is a beta-bonded aldoglycoside radical selected from the group consisting of 1'-aldopentosidyl, 1'-aldohexosidyl, mono-deoxygenated 1'-aldopentosidyl, and mono-deoxygenated 1'-aldohexosidyl and their O-substituted lower alkyl, lower alkanoyl, benzyl and benzoyl derivatives wherein an O-substituent, if present on one oxygen, is present on all available ring substituent oxygens; the tautomers of said isoxanthopterin derivative; and the pharmaceutically acceptable salts of said isoxanthopterin derivative; and maintaining said contact for a time period sufficient for said leucocytes to modulate their immune responses.
 16. The method according to claim 15 wherein the structure of said isoxanthopterin derivative conforms to the formula ##STR11##
 17. The method according to claim 15 wherein R₁ is selected from the group consisting of hydrogen, methyl and carboxy.
 18. A method of enhancing the secretion of antibodies to a preselected immunogen comprising the steps of:contacting immunoglobulin-producing cells with a composition containing a diluent amount of a physiologically tolerable carrier admixed with (a) an effective amount of a preselected immunogen to induce secretion of said antibodies and (b) an adjuvant amount of a isoxanthopterin derivative whose structure conforms to that of the formula ##STR12## wherein R₁ is a radical selected from the group consisting of hydrogen, lower alkyl, hydroxy lower alkyl, polyhydroxy lower alkyl, phenyl, phenyl-lower alkyl, lower alkyl phenyl, lower alkoxy phenyl, halophenyl, trifluoromethyl phenyl, hydroxy, oxo, lower alkoxy, phenyl-lower alkoxy, halo, mercapto, thioxo, lower alkylthio, lower alkyloylthio, phenyl-lower alkylthio, lower alkanoyl, carboxy, lower alkoxy carbonyl, lower alkylcarboxy, lower alkylene lower alkylcarboxylate, lower alkoxy lower alkyl carbonyl, and carboxamido and lower alkyl carboxamido in which the carboxamido group has the formula CONR₃ R₄ wherein R₃ and R₄ are the same or different and are selected from the group consisting of hydrogen and lower alkyl or NR₃ R₄ together form a heterocyclic ring having five or six atoms in the ring; R₂ is a beta-bonded aldoglycoside radical selected from the group consisting of 1'-aldopentosidyl, 1'-aldohexosidyl, mono- deoxygenated 1'-aldopentosidyl, and mono-deoxygenated 1'-aldohexosidyl and their O-substituted lower alkyl, lower alkanoyl, benzyl and benzoyl derivatives wherein an O-substituent, if present on one oxygen, is present on all available ring substituent oxygens; the tautomers of said isoxanthopterin derivative; and the pharmaceutically acceptable salts of said isoxanthopterin derivative; and maintaining said contact for a time period sufficient for said contacted cells to secrete antibodies to said immunogen.
 19. The method according to claim 18 wherein said immunoglobulin-producing cells are primed to said preselected immunogen prior to said contacting step. 